Physicians need rapid and specific histopathologic diagnoses for their cancer patients. For example, open surgical biopsies may contain diagnostic information bearing on intraoperative decisions such as surgical resection vs conservative treatment. But standard rapid diagnosis by frozen section may not discriminate between relevant pathologies. Standard specific diagnosis by immunohistochemistry (IHC) can take days to provide these data. This situation may deny information critical for good decision making at open biopsy. For example, small cell astrocytomas and central nervous system lymphomas are difficult to distinguish on frozen section. Astrocytomas require aggressive resection, but lymphomas are best left unresected. Misdiagnosis could lead to unnecessary resection of eloquent brain or missed aggressive resection of an invasive malignant tumor. A method that rapidly provides relevant diagnostic information will improve clinical care. This project develops nanoprobes that rapidly provide diagnostic histopathologic information from fresh tumor biopsies. Aptamers are an emerging class of polynucleotides easily engineered to identify specific molecular targets. Recent methods let aptamers switch conformation when they bind a target, and this change can provide imaging contrast. Our project aims to generate switchable aptamer contrast agents that detect clinically relevant extracellular and intracellular IHC targets Aim 1 develops a switchable aptamer that rapidly detects the extracellular marker for human central nervous system lymphoma in fresh xenograft tissue biopsies. Aim 2 develops a switchable aptamer against the intracellular astrocytoma marker glial fibrillary acid protein (GFAP). We plan to rapidly deliver the GFAP-specific aptamer to intracellular space using a DNA tetrahedron nanostructure. Both aims optimize rapid aptamer-based labeling of xenograft biopsies. This project develops switchable aptamers as contrast agents for rapid IHC-quality labeling. Our future plan is to develop a clinical trial evaluating aptamer-based diagnostics on intraoperative decision-making during initial craniotomy for brain diagnosis and/or resection. This proposal brings together physicians and scientists from the Barrow Neurological Institute (BNI) and Arizona State University's (ASU) Biodesign Institute. BNI is the largest Neurosurgery residency program in North America and cares for one of the largest volumes of brain tumor patients in the United States. BNI has been a leader in advancing fluorescence imaging tools for brain tumor imaging, and is the only North American center with an advanced Zeiss LSM710/5LiveDUO confocal microscope situated in the pathology department. The Biodesign Institute has been a leader in developing DNA nanotechnology, and its Center for Molecular Design and Biomimetics is innovating the use of DNA as structural material. This collaboration between BNI and the Biodesign Institute will develop an innovative class of nanomolecules for the rapid diagnosis, and improved care of cancer patients.